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Proc. R. Soc. B (2010) 277, 423–427
doi:10.1098/rspb.2009.1378
Published online 14 October 2009
Organic preservation of fossil musculature
with ultracellular detail
Maria McNamara1,*, Patrick J. Orr1, Stuart L. Kearns2, Luis Alcalá3,
Pere Anadón4 and Enrique Peñalver-Mollá5
1
UCD School of Geological Sciences, University College Dublin, Belfield, Dublin 4, Republic of Ireland
2
Department of Earth Sciences, University of Bristol, Wills Memorial Building,
Queen’s Road, Bristol BS8 1RJ, UK
3
Fundación Conjunto Paleontológico de Teruel-Dinópolis, AvenidaSagunto s/n 44002, Teruel, Aragón, Spain
4
Consejo Superior de Investigaciones Cientı́ficas, Institut de Ciències de la Terra ‘Jaume Almera’,
Lluı́s Solé i Sabarı́s s/n 08028, Barcelona, Spain
5
Museo Geominero, Instituto Geológico y Minero de España, C/ Rı́os Rosas 23, 28003 Madrid, Spain
The very labile (decay-prone), non-biomineralized, tissues of organisms are rarely fossilized. Occurrences
thereof are invaluable supplements to a body fossil record dominated by biomineralized tissues, which
alone are extremely unrepresentative of diversity in modern and ancient ecosystems. Fossil examples of
extremely labile tissues (e.g. muscle) that exhibit a high degree of morphological fidelity are almost invariably replicated by inorganic compounds such as calcium phosphate. There is no consensus as to whether
such tissues can be preserved with similar morphological fidelity as organic remains, except when
enclosed inside amber. Here, we report fossilized musculature from an approximately 18 Myr old salamander from lacustrine sediments of Ribesalbes, Spain. The muscle is preserved organically, in three
dimensions, and with the highest fidelity of morphological preservation yet documented from the fossil
record. Preserved ultrastructural details include myofilaments, endomysium, layering within the sarcolemma, and endomysial circulatory vessels infilled with blood. Slight differences between the fossil
tissues and their counterparts in extant amphibians reflect limited degradation during fossilization.
Our results provide unequivocal evidence that high-fidelity organic preservation of extremely labile tissues
is not only feasible, but likely to be common. This is supported by the discovery of similarly preserved
tissues in the Eocene Grube Messel biota.
Keywords: exceptional faunas; taphonomy; muscle; organic preservation; biomolecules
1. INTRODUCTION
The non-biomineralized, decay prone, tissues of organisms are preserved in the fossil record either as organic
remains or via replication in authigenic minerals (Allison &
Briggs 1991; Briggs 2003). Recalcitrant (decay resistant) non-biomineralized tissues, such as cuticles, are
often preserved as organic remains, and, occasionally,
retain some of their original biomolecules (Stankiewicz
et al. 1997; Briggs 1999). However, almost all unequivocal examples of more labile (decay prone) metazoan
tissues, e.g. musculature, are preserved in authigenic
minerals; these replicate tissue structure with varying
degrees of fidelity (depending on the mineral phase and
the timing of mineralization relative to decay). Organic
preservation of tissues inside amber (Poinar & Hess
1982; Grimaldi et al. 1994) can result in retention of
cellular ultrastructures (Henwood 1992), but is rare and
reflects a suite of extremely unusual circumstances.
Charcoalification can preserve labile tissues, but has, to
date, been recorded only in fossil plants (Crepet et al.
1992; Edwards & Axe 2004). Debate continues over the
significance of the organically preserved structures
recovered by Schweitzer et al. (2005) after acid dissolution of samples of dinosaur bone. These structures
were interpreted first as blood vessels and osteocytes largely on the basis of their general morphological similarity
to examples of these in the extant ostrich. The fossil
structures, however, have been reinterpreted as the
remains of recent biofilms that lined, but did not infill
completely, voids inside the bone, thus generating
hollow structures with a similar geometry to blood vessels
and osteocytes (Kaye et al. 2008). Further, the robustness
of the original biochemical analyses that purported to
identify collagen peptide sequence fragments in the
fossil structures (Asara et al. 2007; Schweitzer et al.
2007) has been much debated (Asara & Schweitzer
2008; Asara et al. 2008; Buckley et al. 2008; Organ
et al. 2008; Pevzner et al. 2008; see also Schweitzer
et al. 2009). Suggestions that labile biomolecules are
likely to survive over geological time scales when organisms are encapsulated within natural resins (Poinar et al.
1996) have also been questioned (Stankiewicz et al.
1998). Sulphur-rich organic remains recovered from
inside the bones of fossil frogs hosted within approximately
10 Myr old (Miocene) lacustrine sediments that crop out
near Libros, northeast Spain, were interpreted as the first
(and to date only) examples of fossilized bone marrow
(McNamara et al. 2006). Others, however, have been
more equivocal in their interpretation of these structures
* Author for correspondence ([email protected]).
Electronic supplementary material is available at http://dx.doi.org/10.
1098/rspb.2009.1378 or via http://rspb.royalsocietypublishing.org.
Received 31 July 2009
Accepted 22 September 2009
423
This journal is q 2009 The Royal Society
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424 M. McNamara et al.
Organically preserved muscle
(Stokstad 2006). There is therefore no consensus as to
whether the preservation of very labile non-biomineralized
tissues as organic remains is possible other than via encapsulation within natural resins (amber, copal). Confirming
this is a prerequisite to any investigation of the processes
responsible, including any systematic analysis of the fate
of the original biomolecules under different diagenetic conditions. Herein, we describe, to our knowledge, the first
record of organically preserved musculature including its
sedimentological context. The muscle’s gross morphology
resembles that of an extant analogue, but this, alone, is not
the basis for our conclusion. Remarkably, despite some
degradation before fossilization, diagnostic macromolecular
ultrastructural features have been retained.
2. MATERIAL AND METHODS
The fossilized musculature is from the trunk of a specimen
(Museo Nacional de Ciencias Naturales, Madrid (MNCN)
12555) of the salamander Chelotriton sp. from the Lower- to
Middle Miocene (Agustı́ et al. 1988) Ribesalbes Lagerstätte,
near Castellón de la Plana, northeast Spain. This deposit,
hosted within thermally immature, sulphur-rich oil shales
(kerogen Type I-S: atomic Sorg/C . 0.04; atomic H/C .
1.5; Sinninghe Damsté et al. 1993), yields abundant exceptionally preserved plants and insects. Rare amphibians are
preserved as articulated skeletons enclosed in a thin, black
carbonaceous layer that defines the outline of the soft tissues
(figure 1a). Specimen 12555 was recovered during commercial mining of the oil shales at the beginning of the twentieth
century and does not appear to have been treated or prepared
prior to our analysis.
(a) Fossilized muscle tissue
Samples of muscle tissue identified under a binocular microscope were picked from the specimen using sterile scalpels
and needles. For scanning electron microscopy (SEM),
samples were not prepared further; they were mounted
onto aluminium stubs using double-sided carbon tape, gold
or carbon sputter-coated, and examined with a Hitachi S3500N variable pressure microscope equipped with an
EDAX Genesis energy dispersive spectrometer. Sample conductivity was enhanced by applying small quantities of silver
dag to carbon-coated samples. Backscattered- and secondary
electron images were produced from carbon-coated samples,
and secondary electron images from gold-coated samples.
Observations were made at an accelerating voltage of 15 kV.
For transmission electron microscopy (TEM), samples
were impregnated with TAAB EM resin in an aluminium
mould under vacuum in the following resin/ethanol mixtures,
each for 2 h: 10, 30, 50, 70, 90, 100, 100, 100 per cent resin.
Ultrathin (80–90 nm thick) microtome sections were cut
with a Drukker 2 mm 458 diamond knife, picked up on Cu
grids and allowed to dry. Selected sections were stained
with 2 per cent uranyl acetate in water for 20 min, washed
with distilled water and further stained with lead citrate
(0.01 g lead citrate in 10 ml water and 0.1 ml 10 M
NaOH) for 10 min. Grids were washed again with distilled
water, allowed to dry and examined using a JEOL
2000TEMSCAN operating at 80 kV with an objective aperture of 10 mm diameter. Differences in tone (electron
contrast) in transmission electron micrographs indicate
relative differences in atomic number between features: the
darker the feature, the higher the atomic number of its
Proc. R. Soc. B (2010)
components. Contrast is calibrated automatically by the
camera detector and thus subtle differences in electron contrast between features may not be apparent if an extremely
electron-dense feature is in the field of view.
(b) Modern muscle tissue
Muscle tissue from the extant salamander Ambystoma
mexicanum supplied in 100 per cent ethanol (after primary
fixation in gluataraldehyde) was treated as follows: immersion in osmium tetroxide (60 min), buffer (a mixture of
sodium dihydrogen phosphate and disodium hydrogen
phosphate) (10 min), 70 per cent ethanol (10 min (twice)),
90 per cent ethanol (10 min (twice)) and 100 per cent ethanol (20 min (twice)). For TEM, fixed and dehydrated
samples were prepared further as follows: immersion in propylene oxide (15 min (twice)), 50 : 50 propylene oxide:
TAAB EM (60 min) and 100 per cent resin (120 min at
378C). Resin impregnation was conducted under a vacuum
of 86–90 kPa using a Buehler Vacuum Impregnation Unit.
Samples were then placed in an aluminium mould with
fresh 100 per cent resin and polymerized under vacuum for
18 h at 608C. Ultrathin (80– 90 nm thick) sections were
placed on Cu grids, stained with uranyl acetate and lead
citrate and examined as for the fossil material. SEM samples
were prepared and analysed as for the fossil material.
3. FOSSIL MUSCLE
Hypaxial skeletal muscle fibres are preserved immediately
adjacent to the thoracic vertebrae (figure 1b,c). As in
modern salamanders (Brainerd & Azizi 2005), the fibres
are closely and regularly spaced, parallel and stacked in
sheets separated by endomysium (figure 1d –g). Hollow
tubes with circa 3 mm thick walls orientated orthogonally
to the axis of some fibres (figure 1d, arrow) represent sections through vascular structures, probably arterioles and
venules. Each fibre is straight, unbranched, 15 – 25 mm
wide and exhibits a chevron-shaped pattern of elongate
cracks, one set on either side of the medial axis
(figure 1d,e). In transverse section, each fibre is square
to rectangular in outline, has concave sides and an irregular, circular to oval-shaped, central void (figure 1f ).
These voids, and the cracks, are attributed to diagenetic
shrinkage of the fibres, probably via condensation of the
cytosol. In stained transverse TEM sections, myofilaments are apparent as two sets of densely packed,
electron-dense, ‘spots’ (figure 2a): spots of one set are
larger (10– 14 nm diameter as opposed to 5 –8 nm diameter) and more electron dense. The spots are sections
through thick (myosin-rich) and thin (actin-rich) myofilaments; the presence of both indicates the plane of
sectioning has passed through the A-band of a fibre.
The structure in the fossil material is slightly less ordered
than the regular hexagonal arrangement found in its
modern counterpart (compare figure 2a and b); the loss
of fidelity probably originated during initial, limited,
decay before fossilization. Myofibrils are not apparent in
the fossil material, even in stained sections, but this is
not unexpected. In many extant amphibians, the trunk
muscles are tonic, and the myofilaments are weakly or
not bundled into myofibrils (Gans 1981). The sarcolemma is typically 800– 1200 nm thick, slightly greater
than in extant salamanders (650 – 850 nm thick) (compare figure 2c,e with 2d, f ). This difference may be
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Organically preserved muscle
(a)
M. McNamara et al. 425
(b)
(c)
(d)
(e)
(f)
(g)
Figure 1. Organically preserved musculature in a fossil salamander. (a) Chelotriton sp. (MNCN 12555) with an outline of
soft tissues defined by a carbonaceous layer. Scale bar, 10 mm. (b) Musculature in situ adjacent to vertebrae and (c) a freshly
fractured surface through a sample. Scale bar, 1 mm. (d, arrow) SEM images of muscle illustrating chevron pattern of cracks,
hollow blood vessel in transverse section and (e, arrow) separation of sarcolemma from sarcoplasm. Scale bars: (d ) 100 mm and
(e) 30 mm. ( f ) Semithin transverse section through the muscle with (g) explanatory drawing, showing the arrangement of fibres
(dark grey) in parallel sheets separated by endomysium (light grey); arrow in ( f ), blood vessel in the transverse section.
( f ) Scale bar, 20 mm.
real; alternatively, the thickness of the sarcolemma in the
fossil muscle may have increased during minor postmortem separation of collagen fibres (Taylor et al.
1995). Internally, the sarcolemma can be either structureless with a very thin, relatively electron-dense, margin
(figure 2c), or multi-layered (figure 2e); both conditions
occur in extant material (figure 2d, f ). The multi-layered
structure is more apparent where the sarcolemma has
detached from the interior of the fibre and is convoluted;
in such examples, the sarcolemma often separates further
into a series of layers (figure 2e), a characteristic of the
initial stages of its decay (Taylor et al. 1995).
Adjacent sheets of muscle fibres are separated by a thin
(2 – 6 mm thick) endomysium. The endomysium is often
laminated and can separate into its component layers
(figure 2g). Endomysial circulatory vessels (4 –10 mm in
diameter and circular in cross section) (figure 2g) can
be filled with a friable, highly electron-dense, material
(figure 2g, inset). This infill is extremely unstable under
the electron beam and usually disintegrates during examination, producing resin-free voids. The infill occurs only
Proc. R. Soc. B (2010)
within these circulatory vessels, not other void spaces, and
has been observed in unstained sections. Collectively,
these observations confirm the infill is of biological
origin, not a diagenetic infill of void space or an artefact
of sample preparation; circulatory vessels in modern salamander muscle are infilled with blood (figure 2h). The
fossil blood is structureless, and there is no evidence for
spherical features, e.g. red blood cells (most purported
fossil examples of which have been demonstrated to be
pyrite framboids, Martill & Unwin 1997). The composition
of the fossil blood is unknown; its high electron density indicates that it is unlikely to be entirely carbonaceous. EDX
spectra of the musculature exhibit peaks for only C and
S. The musculature is therefore not replicated in authigenic
minerals. It is, at least primarily, the diagenetically altered
remains of the original organic tissues.
4. WIDER IMPLICATIONS
The detail revealed by TEM imaging unequivocally identifies the organic remains as fossilized musculature from
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426 M. McNamara et al.
(a)
Organically preserved muscle
(c)
(e)
(g)
sp
sp
sl
r
sl
l
r
(b)
(d)
(f)
(h)
Figure 2. (a,c,e,g) TEM images of transverse sections of skeletal muscle tissue from MNCN 12555 and (b,d,f,h) corresponding
features in the extant salamander A. mexicanum. Images except (c), (e) and (g) are from stained sections. (a,b) Sarcoplasm
showing myofilaments in the transverse section at A-band. (c,d ) Margin of fibre showing sarcolemma (sl) attached to sarcoplasm (sp); inset in (d ) shows local thickening of sarcolemma. (e, f ) Margin of fibres showing sarcolemma (sl) detached
from sarcoplasm (sp). (g,h) Endomysial circulatory vessels surrounded by endomysium. Inset in (g) shows electron-dense, friable, solidified blood residue within the circulatory vessel. l, lumen of capillary; r, resin. Scale bars (a,b) 50 nm and 2 mm for all
other images.
the salamander itself. This therefore confirms, for the first
time, to our knowledge, that the high-fidelity fossilization
of extremely decay-prone tissues as organic remains is not
only feasible but can occur in the absence of protective
encapsulating agents such as bone (in the case of the
bone marrow, McNamara et al. 2006) and amber.
Preservation of the musculature as a sulphur-rich
organic residue is attributed to sulphurization of organic
molecules within the tissue (McNamara et al. 2006).
Sulphurization of organic matter is a common diagenetic
phenomenon and has been documented in various carbonate, evaporite and siliceous ooze-dominated, marine,
and non-marine environments (Killops & Killops 2005);
this includes environmental contexts known to host
exceptional faunas (Sinninghe Damsté et al. 1993; de
las Heras et al. 2003). Further, this mode of exceptional
preservation, articulated skeletons encased in a carbonaceous layer that defines the body outline, characterizes
other Mesozoic and, especially, Cenozoic lacustrinehosted biotas from Europe and elsewhere; examples
include the Messel (Eocene, Germany) (Franzen 1990),
Enspel (Oligocene, Germany) (Toporski et al. 2002),
Bechlejovice (Oligocene, Czech Republic) (Roček 2003)
and Shiobara (Pleistocene, Japan) (Allison et al. 2008)
biotas. We therefore predict that careful examination of
specimens will confirm that high-fidelity organic preservation of labile soft tissues is relatively common in the
fossil record, particularly in lacustrine settings. This is
supported by the preliminary investigation of organically
preserved tissues from several taxa from the Messel
biota (see the electronic supplementary material). Fauna
from such biotas are prime targets for the recovery of
other examples of high-fidelity, organically preserved
soft tissues, and, potentially, labile biomolecules.
We thank Cormac O’Connell and Dave Cottell for assistance
with TEM analyses, Eric Callaghan for assistance with the
preparation of histological sections, Seán Burke for field
assistance, the Generalitat Valenciana for access to the
Ribesalbes site, Adam Franssen and Neil Shubin for
providing specimens of A. mexicanum, and Dra Begoña
Proc. R. Soc. B (2010)
Sánchez (MNCN, Madrid) for access to the fossil
specimen. The study was funded by Enterprise Ireland
Basic Research grant SC/2002/138 to P.J.O. We thank
Dr Dave Martill and an anonymous reviewer for their
comments.
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